Recently, mesothelin has been identified as an antigen specific to 95% of ovarian cancers, making it a potentially valuable immunotherapeutic target. We have developed a mesothelin-expressing pre-clinical tumor model, WF-3, that will allow us to test DNA vaccine strategies for control of ovarian cancer. One major hurdle for cancer vaccine development is the issue of immunological tolerance to self antigens. The employment of heteroclitic CTL peptides from tumor-associated antigens has been recognized as an important strategy to break tolerance. We have recently demonstrated that intradermal vaccination with DNA encoding single chain trimers of MHC class I with CTL peptides derived from a murine melanoma self antigen can bypass antigen processing and lead to stable presentation of peptides. Our DNA vaccine can break tolerance to the self antigen and elicit antigen-specific antitumor immunity in vaccinated mice. Immunization resulted in an increased number of antigen-specific CD8+ T cells and tumor rejection and tumor growth suppression after a lethal challenge of B16 murine melanoma cells in C57BL/6 mice. Thus, in the current proposal, we plan to test if a DNA vaccine encoding single chain trimers of MHC class I with heteroclitic CTL peptides derived from mesothelin will facilitate the breaking of mesothelin tolerance and lead to mesothelin-specific antitumor effects against a mesothelin-expressing tumor model in vivo. Specifically, we plan to characterize the mesothelin-specific CD8+ T cell immune response and determine the antitumor effect against mesothelin-expressing tumors in C57BL/6 mice vaccinated with the various DNA constructs encoding single chain trimers of MHC class I linked to a heteroclitic CTL peptide derived from mesothelin. We will compare our best DNA vaccine with the best vaccine from Project 1 in our WF-3 preclinical tumor model. The best vaccine strategy will then be evaluated in combination with the most effective immune modulating agents from Projects 3 and 4. The successful implementation of the proposed studies will provide a foundation for further clinical translation.